Under the influence of the thymic environment, which is comprised of bone marrow derived cells and epithelium, several important aspects of T cell differentiation occur, including antigen receptor expression, establishment of MHC-restricted antigen recognition, and self-tolerance. Understanding the contributions of thymic epithelium (TE) to T cell differentiation has been hampered by a lack of information regarding thymic epithelium. The proposed work will generate TE cell lines and use these cell lines to characterize TE biochemically and functionally. Cell lines representative of cortical and medullary TE will be examined for their ability to physically associate with thymocyte subpopulations in vitro, to secrete cytokines affecting thymocyte proliferation/ differentiation, and to present antigen in the context of major histocompatibility complex molecules.These cell lines will be used to generate a panel of monoclonal antibodies specific for cell surface molecules expressed by cortical and medullary TE populations. Anti-TE antibodies will be used to immunochemically characterize the cell surface molecules expressed by different populations of thymic epithelium cells and to identify molecules unique to cortical and medullary thymic epithelial cells. Immunochemical studies of these molecules will be complemented by immunoelectron microscopic studies to precisely define their in situ distribution within the thymus. Finally, by testing the effects of these anti-epithelial antibodies on thymic function in vivo and vitro, the participation of these epithelial cell surface molecules in the process of T cell differentiation will be assessed. These studies will utilize multi-parameter flow cytometry to analyze thymocyte populations differentiating in the presence of anti-TE monoclonal antibodies specific for cortical or medullary TE. The significance of the proposed work is that it will define the thymic environment in terms of the epithelial cell surface and/or cytokines that collectively constitute the thymic environment. Characterization of the constituent components of the thymic environment will lead to a clearer understanding of the process of T cell differentiation and may lead to the design of therapeutic approaches to enhance thymic function.